Printing molecular library arrays

ABSTRACT

A method and apparatus for selectively applying a print material onto a substrate for the synthesis of an array of oligonucleotides at selected regions of a substrate. The print material includes a barrier material, a monomer sequence, a nucleoside, a deprotection agent, a carrier material, among other materials. The method and apparatus also relies upon standard DMT based chemistry, and a vapor phase deprotection agent such as solid TCA and the like.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the synthesis and placement ofmaterials at known locations. In particular, one embodiment of theinvention provides a method and associated apparatus for the selectiveapplication of an array of oligonucleotides on a substrate by way ofstandard dimethoxytrityl (DMT) based chemistry. The invention may beapplied in the field of preparation of an oligomer, a peptide, a nucleicacid, an oligosaccharide, a phospholipid, a polymer, or a drug congenerpreparation, especially to create sources of chemical diversity for usein screening for biological activity.

[0002] Industry utilizes or has proposed various techniques tosynthesize arrays of oligonucleotides. One such technique is the use ofsmall rubber tubes as reaction chambers to make up a single dimensionalarray by the sequential addition of reagents. This technique hasadvantages by the use of standard DMT based chemistry. However, alimitation with resolution often exists with such technique. Typicallythe smallest cell size is about 1 millimeter in dimension. This methodalso does not enable the synthesis of a sufficiently large number ofpolymer sequences for effective economical screening. A furtherlimitation is an inability to form an array of, for example,oligonucleotides at selected regions of a substrate.

[0003] Other representative techniques are described in U.S. Pat. No.5,143,854 and WO93/09668 which is hereby incorporated by reference forall purposes. Such techniques are finding wide use and are consideredpioneering in the industry. In some applications, however, it isdesirable to have alternative techniques and chemistries for synthesisof compound libraries.

[0004] It would be desirable to have a method and apparatus for makinghigh density arrays of oligonucleotides using DMT-based chemistry andother suitable oligonucleotide synthesis chemistries, as is a method andapparatus for conventional phosphoramidite-based synthesis of aspatially defined array of oligomers (e.g., polynucleotides,polypeptides, oligosaccharides, and the like) each having asubstantially predetermined sequence of residues (i.e., polymerizedmonomer units).

SUMMARY OF THE INVENTION

[0005] According to the present invention, a method and apparatus toform an array of polymers, such as oligonucleotides and related polymers(e.g., peptide nucleic acids) at selected regions of a substrate usingconventional linkage chemistries (e.g., standard DMT-basedoligonucleotide synthesis chemistry) is provided. The method andapparatus includes use of selected printing techniques in distributingmaterials such as barrier materials, deprotection agents, base groups,nucleosides, nucleotides, nucleotide analogs, amino acids, imino acids,carrier materials, and the like to selected regions of a substrate. Eachof the printing techniques may be used in some embodiments with, forexample, standard DMT-based chemistry for synthesis of oligonucleotides,and in particular selected deprotecting agents in vapor form.

[0006] In a specific embodiment, the present invention provides a methodof forming polymers having diverse monomer sequences on a substrate. Inan embodiment, the method is used to synthesize oligonucleotides havingpredetermined polynucleotide sequence(s) on a solid substrate, typicallyin the form of a spatially defined array, wherein the sequence(s) of anoligonucleotide is positionally determined. The present method includessteps of providing a substrate with a linker molecule layer thereon. Thelinker molecule layer has a linker molecule and a protective group. Thepresent method also includes a step of applying a barrier layeroverlying at least a portion of the linker molecule layer. The barrierlayer shields the underlying portion from contact with a reagent capableof otherwise reacting with the underlying portion and applied subsequentto application of the barrier layer, thereby substantially precluding apredetermined chemical reaction from occurring on areas of the substrateoverlaid with the barrier material. The applying step forms selectedexposed regions of the linker molecule layer. A step of exposing theselected exposed regions of the linker molecule layer (e.g., regions notoverlaid with the barrier material) to a reagent, typically in vaporphase, and often comprising a deprotecting agent is also included.

[0007] In an alternative specific embodiment, the present methodincludes a method of applying a medium in selected regions of asubstrate. The present method includes steps of providing a substratewith a top surface, and selectively applying a medium having an elementselected from a group consisting of a barrier material, a receptor, adeprotection agent, a monomer group, a carrier material, and anactivator to selected regions of the substrate top surface.

[0008] In an embodiment, the invention provides a method forsynthesizing a spatial array of polymers of diverse monomeric sequence(e.g., such as a collection of oligonucleotides having uniquesequences), wherein the composition (e.g., nucleotide sequence) of eachpolymer is positionally defined by its location in the spatial array. Ingeneral, the method employs a masking step whereby a spatiallydistributed barrier material is applied to a substrate to block at leastone step of a monomer addition cycle from occurring on a portion of thesubstrate overlaid by the barrier material. The method comprisesapplying a barrier material to a first spatially defined portion of asubstrate, said substrate optionally also comprising a layer of linkermolecules and/or nascent polymers (e.g., nascent oligonucleotides),whereby the barrier material overlaying said first spatially definedportion of said substrate shields the underlying portion from contactwith a subsequently applied reagent capable of otherwise reacting withthe underlying portion and necessary for a complete monomer additioncycle whereby a monomer unit is covalently linked to a nascent polymeror linker, thereby substantially precluding a chemical reaction fromoccurring on said first spatially defined portion which is overlaid withthe barrier material and providing a remaining unshielded portion ofsaid substrate (i.e., portion(s) not overlaid with the barrier material)available for contacting said subsequently applied reagent andundergoing said chemical reaction necessary for a complete monomeraddition cycle (i.e., polymer elongation). The subsequently appliedreagent is typically a monomer (e.g., nucleotide, nucleoside, nucleosidederivative, amino acid, and the like), a deprotecting agent for removingprotecting group(s) which block polymer elongation (e.g., removal of DMTgroups by acid hydrolysis), a coupling agent (e.g., phosphoramidites,such as cyanoethyl phosphoramidite nucleosides), a capping agent (e.g.,acetic anhydride and 1-methylimidazole), and/or an oxidation agent(e.g., iodine; such as in iodine:water:pyridine:tetrahydrofuranmixture). The method further provides that, subsequent to theapplication of the barrier material, the reagent(s) is/are applied andpermitted to chemically react with the unshielded portion of thesubstrate for a suitable time period and under suitable reactionconditions. Following reaction of the unshielded portion with thereagent(s), monomer addition is completed and the barrier material isremoved (not necessarily in that order), resulting in a monomer additionto polymer(s) in the unshielded portion of the substrate and substantiallack of monomer addition to polymer(s) in the shielded portion of thesubstrate, during said monomer addition cycle.

[0009] In an embodiment, the masking step, wherein,a barrier material isapplied to a spatially defined portion of the substrate and used toshield said spatially defined portion to block a monomer addition cycleon said spatially defined portion, is employed repetitively. A firstbarrier mask is applied to overlay a first spatially defined portion ofa substrate creating: (1) a first shielded portion overlain by saidbarrier mask, and (2) a first unshielded portion comprising the portionof the substrate not overlain by said barrier mask. The application ofthe first barrier mask is followed by completion of a first monomeraddition cycle, whereby a monomer unit is covalently added to the firstunshielded portion to extend or initiate a nascent polymer bound to saidsubstrate, typically covalently, and whereby said first monomer additioncycle substantially fails to result in addition of a monomer unit tonascent polymers in the first shielded portion. The first barrier maskis removed, concomitant with, prior to, or subsequent to the completionof said first monomer addition cycle, and one or more subsequent cyclesof applying a subsequent barrier mask, which may overlay subsequentshielded portions which is/are spatially distinct from said firstshielded portion, and performing at least one subsequent monomeraddition cycle(s) followed after each cycle by barrier removal, andoptionally, reapplication of a barrier mask and initiation of a furthermonomer addition cycle until polymers of a predetermined length (numberof incorporated monomer units) are produced.

[0010] In an aspect of the invention, a repetitive masking/synthesisprocess can be comprised of the following steps:

[0011] (1) application of barrier material to substrate having areactive surface capable of covalently bonding to a monomer unit orreacting with a deprotecting agent or other reagent necessary forcompletion of a monomer addition cycle, said reactive surface beingderivatived with a linker and/or a monomer unit or nascent polymer(e.g., a 3′-linked nucleoside or 3′-linked polynucleotide), wherein saidbarrier material covers a portion of said reactive surface creating acovered portion, said covered portion being a shielded portion and beingsubstantially incapable of reacting with a monomer unit or reagentnecessary for completion of a monomer addition cycle, and the remainingportion of the substrate being an unshielded portion capable of reactingwith a monomer unit or reagent necessary for completion of a monomeraddition cycle;

[0012] (2) contacting the substrate with reagents necessary forcompletion of a monomer addition cycle, wherein a monomer unit iscovalently attached to the reactive surface of the substrate (e.g., alinker, a 3′-linked nucleoside, or 3′-linked nascent polynucleotide) inan unshielded portion;

[0013] (3) removing the barrier material; and

[0014] (4) repeating steps 1, 2, and 3 from 0 to 5000 cycles, preferablyfrom 2 to 250 cycle, more usually from 4 to 100 cycles, and typicallyfrom about 7 to 50 cycles, until a predetermined polymer length isproduced on a portion of the substrate. The pattern of barrier materialapplied in each cycle may be different that the prior or subsequentcycle(s), if any, or may be the same. Often, in step (2), at least onereagent necessary for completion of a monomer addition cycle is appliedin vapor phase.

[0015] In an embodiment of the invention is provided a substrate havinga spatial array of polymers of predetermined length produced by themethod described supra.

[0016] In one aspect of the invention is provided a method for applyinga barrier material or reagent necessary for a monomer addition cycle toa substrate, said method comprising transferring the barrier material orreagent as a charged droplet by electrostatic interaction, such as, forexample, in an inkjet or bubble jet print head or similar device. In anembodiment, the barrier material or reagent is suitable for use inpolynucleotide (oligonucleotide) synthesis. In an embodiment, thesubstrate is a silicon or glass substrate or a charged membrane (e.g.,nylon 66 or nitrocellulose).

[0017] An aspect of the invention provides a method for synthesizingpolynucleotides on a substrate, said method comprising application of atleast one reagent necessary for addition of a nucleotide to a nascentpolynucleotide or linker molecule bound to a substrate, wherein saidapplication is performed with the reagent present substantially in vaporphase.

[0018] A further understanding of the nature and advantages of thepresent invention may be realized by reference to the latter portions ofthe specification and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1-3 illustrate simplified cross-sectional views of asubstrate being processed according to the present invention;

[0020] FIGS. 4-13 illustrate selected printing techniques according tothe present invention;

[0021]FIG. 14 illustrates a simplified cross-sectional view of anapparatus used to achieve local selectivity;

[0022]FIG. 15 illustrates a jig used for contacting a mask to asubstrate without smearing;

[0023]FIG. 16 is a photograph of a fluorescent image of a fluoreprimedworkpiece that was selectively shielded from liquid deprotection by alacquer;

[0024]FIG. 17 is a photograph of dots of uncured epoxy and pump oiloverlying a workpiece;

[0025]FIG. 18 illustrates a SEM photograph of a liquid uncured epoxypattern on a glass workpiece;

[0026]FIG. 19 illustrates a SEM photograph of a 100 micron resolutionsample with an epoxy barrier pattern;

[0027]FIG. 20 illustrates a SEM photograph of a 75 micron resolutionsample with an epoxy barrier pattern;

[0028]FIG. 21 is a photograph of a fluorescent.pattern from vapordeprotection through an uncoated silicon stencil mask;

[0029]FIG. 22 is a close-up version of the photograph of FIG. 21;

[0030]FIG. 23 is a photograph of an epoxy paint pattern transferred froma nickel grid;

[0031]FIGS. 24 and 25 are photographs of fluorescent images resultingfrom vapor phase deprotection through an epoxy pattern;

[0032]FIG. 26 illustrates a 2×2 array of oligonucleotides formed bymasking out deprotection agents after A (vertical mask) and a first T inthe synthesis of 3′-CGCATTCCG;

[0033]FIG. 27 is a scanned output of an array after hybridizing with 10nM target oligonucleotide 5′-GCGTAGGC-fluorescein for 15 minutes at 15C;

[0034]FIGS. 28 and 29 are scanned outputs after hybridizing to anewly-made sample of the same target sequence of FIGS. 26 and 27;

[0035]FIG. 30 is an array of same oligos as in FIGS. 26 and 27 made bydisplacing the reaction chamber when added bases A and the first T inthe sequence 3′-CGCATTCCG;

[0036]FIGS. 31 and 32 illustrate scanned outputs after hybridizing with10 nM 5′-GCGTAGGC-fluorescein.

DESCRIPTION OF THE SPECIFIC EMBODIMENT

[0037] Glossary

[0038] The following terms are intended to have the following generalmeanings as they are used herein:

[0039] 1. Ligand: A ligand is a molecule that is recognized by aparticular receptor. Examples of ligand that can be investigated by thisinvention include, but are not restricted to, agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes, hormones(e.g., opiates, steroids, etc.), hormone receptors, peptides, enzymes,enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides,nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

[0040] 2. Monomer: A member of the set of small molecules which are orcan be joined together to form a polymer. The set of monomers includesbut is not restricted to, for example, the set of common L-amino acids,the set of D-amino acids, the set of synthetic and/or natural aminoacids, the set of nucleotides and the set of pentoses and hexoses. Theparticular ordering of monomers within a polymer is referred to hereinas the “sequence” of the polymer. As used herein, monomers refers to anymember of a basis set for synthesis of a polymer, which include forexample and not limitation, polynucleotides, polypeptides, and smallmolecules such as benzodiazepines, β-turn mimetics, andprotoprostaglandins, among others. For example, dimers of the 20naturally occurring L-amino acids form a basis set of 400 monomers forsynthesis of polypeptides. Different basis sets of monomers may be usedat successive steps in the synthesis of a polymer. Furthermore, each ofthe sets may include protected members which are modified aftersynthesis. The invention is described herein primarily with regard tothe preparation of molecules containing sequences of monomers such asamino acids, but could readily be applied in the preparation of otherpolymers. Such polymers include, for example, both linear and cyclicpolymers of nucleic acids, polysaccharides, phospholipids, and peptideshaving either α-, β-, OR ω-amino acids, heteropolymers in which a knowndrug is covalently bound to any of the above, polynucleotides,polyurethanes, polyesters, polycarbonates, polyureas, polyamides,polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides,polyacetates, or other polymers which will be apparent upon review ofthis disclosure. Such polymers are “diverse” when polymers havingdifferent monomer sequences are formed at different predefined regionsof a substrate. Methods of cyclization and polymer reversal of polymersare disclosed in application Ser. No. 07/796,727 filed Nov. 22, 1991(now U.S. Pat. No. 5,242,974 issued Sep. 7, 1993, entitled “POLYMERREVERSAL ON SOLID SURFACES,” incorporated herein by reference for allpurposes. One set of polymers is polynucleotides and peptide nucleicacids.

[0041] 3. Peptide: A polymer in which the monomers are alpha amino acidsand which are joined together through amide bonds, alternativelyreferred to as a polypeptide. In the context of this specification itshould be appreciated that the amino acids may be the L-optical isomeror the D-optical isomer. Peptides are often two or more amino acidmonomers long, and often more than 20 amino acid monomers long. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber andFreidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30:1229, which are incorporated herein by reference) and are oftendeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics have one or more peptidelinkages optionally replaced by a linkage selected from the groupconsisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods known in the art andfurther described in the following references: Spatola, A. F. in“Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,” B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F.,Vega Data (March 1983), Vol. 1, Issue 3, “Peptide BackboneModifications” (general review); Morley, J. S., Trends Pharm Sci (1980)pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res(1979) 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola, A. F. et al., Life Sci(1986) 38:1243-1249 (—CH₂—S); Hann, M. M., J Chem Soc Perkin Trans I(1982) 307-314 (—CH—CH—, cis and trans); Almquist, R. G. et al., J MedChem (1980) 23:1392-1398 (—COCH₂—); Jennings-White, C. et al.,Tetrahedron Lett (1982) 23:2533 (—COCH₂—); Szelke, M. et al., EuropeanAppln. EP 45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH₂—); Holladay, M.W. et al., Tetrahedron Lett (1983) 24:4401-4404 (—C(OH)CH₂—); and Hruby,V. J., Life Sci (1982) 31:189-199 (—CH₂—S—); each of which isincorporated herein by reference. A particularly preferred non-peptidelinkage is —CH₂NH—. Such peptide mimetics may have significantadvantages over polypeptide embodiments, including, for example: moreeconomical production, greater chemical stability, enhancedpharmacological properties (half-life, absorption, potency, efficacy,etc.), altered specificity (e.g., a broad-spectrum of biologicalactivities), reduced antigenicity, and others. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptides(including cyclized peptides) comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem.61: 387, incorporated herein by reference); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

[0042] 4. Receptor: A molecule that has an affinity for a given ligand.Receptors may be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex. Specific examples ofreceptors which can be investigated by this invention include but arenot restricted to:

[0043] a) Microorganism receptors: Determination of ligands which bindto receptors, such as specific transport proteins or enzymes essentialto survival of microorganisms, is useful in a new class of antibiotics.Of particular value would be antibiotics against opportunistic fungi,protozoa, and those bacterial resistant to the antibiotics in currentuse.

[0044] b) Enzymes: For instance, the binding site of enzymes such as theenzymes responsible for cleaving neurotransmitters; determination ofligands which bind to certain receptors to modulate the action of theenzymes which cleave the different neurotransmitters is useful in thedevelopment of drugs which can be used in the treatment of disorders ofneurotransmission.

[0045] c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the development ofvaccines of which the immunogen is based on one or more of suchsequences or led to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto-immunediseases (e.g., by blocking the binding of the “self” antibodies).

[0046] d) Nucleic Acids: Sequences of nucleic acids may be synthesizedto establish DNA or RNA binding sequences. Polynucleotides, whichinclude oligonucleotides, are composed of nucleotides, typically linked5′ to 3′ by a phosphodiester bond or phosphorothiolate bond or the like.The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”. Polynucleotidescan include nucleotides having a variety of bases, including but notlimited to: adenine, thymine, cytosine, guanine, uridine, inosine,deazaguanosine, N²-dimethylguanosine, 7-methylguanosine, N⁶-Δ²isopentenyl-2-methylthioadenosine, 2′-O-methyladenine,2′-O-methylthymine, 2′-O-methylcytosine, 2′-O-methylguanine,pseudouridine, dihydrouridine, 4-thiouridine, and the like.

[0047] e) Catalytic Polypeptides: Polymers, preferably polypeptides,which are capable of promoting a chemical reaction involving theconversion of one or more reactants to one or more products. Suchpolypeptides generally include a binding site specific for at least onereactant or reaction intermediate and an active functionality proximateto the binding site, which functionality is capable of chemicallymodifying the bound reactant. Catalytic polypeptides and others aredescribed in, for example, PCT Publication No. WO 90/05746, WO 90/05749,and WO 90/05785, which are incorporated herein by reference for allpurposes.

[0048] f) Hormone Receptors: For instance, the receptors for insulin andgrowth hormone. Determination of the ligands which bind with highaffinity to a receptor is useful in the development of, for example, anoral replacement of the daily injections which diabetics must take torelieve the symptoms of diabetes, and in the other case, a replacementfor the scarce human growth hormone which can only be obtained fromcadavers or by recombinant DNA technology. Other examples are thevasoconstrictive hormone receptors; determination of those ligands whichbind to a receptor may lead to the development of drugs to control bloodpressure.

[0049] g) Opiate receptors: Determination of ligands which bind to theopiate receptors in the brain is useful in the development ofless-addictive replacements for morphine and related drugs.

[0050] 5. Substrate: A material having a rigid or semi-rigid surface. Inmany embodiments, at least one surface of the substrate will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different polymers with, forexample, wells, raised regions, etched trenches, or the like. Accordingto other embodiments, small beads may. be provided on the surface whichmay be released upon completion of the synthesis. Often, the substrateis a silicon or glass surface, or a charged membrane, such as nylon 66or nitrocellulose.

[0051] 6. Protective Group: A material which is bound to a monomer unitand which may be selectively removed therefrom to expose an active sitesuch as, in the specific example of an amino acid, an amine group. Inthe specific example of a polynucleotide synthesized via phosphoramiditechemistry, a protecting group can be a trityl ether (DMT ether) grouplinked to a nucleotide via a 5′-hydroxyl position.

[0052] 7. Predefined Region: A predefined region is a localized area ona substrate which is, was, or is intended to be used for formation of aselected polymer and is otherwise referred to herein in the alternativeas a “selected” region or simply a “region.” The predefined region mayhave any convenient shape, e.g., circular, rectangular, elliptical,wedge-shaped, etc. In some embodiments, a predefined region and,therefore, the area upon which each distinct polymer sequence issynthesized is smaller than about 1 cm², more preferably less than 1mm², still more preferably less than 0.5 mm², and in some embodimentsabout 0.125 to 0.5 mm². In most preferred embodiments the regions havean area less than about 10,000 μm² or, more preferably, less than 100μm². Within these regions, the polymer synthesized therein is preferablysynthesized in a substantially pure form. A shielded portion orunshielded portion can be a predefined region.

[0053] 8. Substantially Pure: A polymer is considered to be“substantially pure” within. a predefined region of a substrate when itexhibits characteristics that distinguish it from other predefinedregions. Typically, purity will be measured in terms of biologicalactivity or function as a result of uniform sequence. suchcharacteristics will typically be measured by way of binding with aselected ligand or receptor. Preferably the region is sufficiently puresuch that the predominant species in the predefined region is thedesired sequence. According to preferred aspects of the invention, thepolymer is 5% pure, more preferably more than 10% pure, preferably morethan 20% pure, and more preferably more than 80% pure, more preferablymore than 90% pure, more preferably more than 95% pure, where purity forthis purpose refers to the ratio of the number of ligand moleculesformed in a predefined region having a desired sequence to the totalnumber of molecules formed in the predefined region.

[0054] 9. Monomer Addition Cycle: A monomer addition cycle is a cyclecomprising the chemical reactions necessary to produce covalentattachment of a monomer to a nascent polymer or linker, such as toelongate the polymer with the desired chemical bond (e.g., 5′-3′phosphodiester bond, peptide bond, etc.). For example and not to limitthe invention, the following steps typically comprise a monomer additoncycle in phosphoramidite-based oligonucleotide synthesis: (1)deprotection, comprising removal of the DMT group from a 5′-protectednucleoside (which may be part of a nascent polynucleotide) wherein the5′-hydroxyl is blocked by covalent attachment of DMT, such deprotectionis usually done with a suitable deprotection agent (e.g., a protic acid:trichloroacetic acid or dichloroacetic acid), and may include physicalremoval (e.g., washing, such as with acetonitrile) of the removedprotecting group (e.g., the cleaved dimethyltrityl group), (2) coupling,comprising reacting a phosphoramidite nucleoside(s), often activatedwith tetrazole, with the deprotected nucleoside, (3) optionallyincluding capping, to truncate unreacted nucleosides from furtherparticipation in subsequent monomer addition cycles, such as by reactionwith acetic anhydride and N-methylimidazole to acetylate free5′-hydroxyl groups, and (4) oxidation, such as by iodine intetrahydrofuran/water/pyridine, to convert the trivalent phosphitetriester linkage to a pentavalent phosphite triester, which in turn canbe converted to a phosphodiester via reaction with ammonium hydroxide.Thus, with respect to phosphoramidite synthesis of polynucleotides, thefollowing reagents are typically necessary for a complete monomeraddition cycle: trichloroacetic acid or dichloroacetic acid, aphosphoramidite nucleoside, an oxidizing agent, such as iodine (e.g.,iodine/water/THF/pyridine), and optionally N-methylimidazole forcapping.

[0055] 10. Specific hybridization is defined herein as the formation ofhybrids between a probe polynucleotide (e.g., a polynucleotide of theinvention which may include substitutions, deletion, and/or additions)and a specific target polynucleotide (e.g., an analyte polynucleotide)wherein the probe preferentially hybridizes to the specific targetpolynucleotide and substantially does not hybridize to polynucleotidesconsisting of sequences which are not substantially identical to thetarget polynucleotide. However, it will be recognized by those of skillthat the minimum length of a polynucleotide required for specifichybridization to a target polynucleotide will depend on-several factors:G/C content, positioning of mismatched bases (if any), degree ofuniqueness of the sequence as compared to the population of targetpolynucleotides, and chemical nature of the polynucleotide (e.g.,methylphosphonate backbone, phosphorothiolate, etc.), among others.

[0056] General

[0057] The present invention provides for the use of a substrate with asurface. In preferred embodiments, linker molecules are provided on asurface of the substrate. The purpose of the linker molecules, incertain embodiments, is to facilitate receptor recognition of thesynthesized polymers. In preferred embodiments, the linker moleculeseach include a protection group. A layer of barrier material may beapplied to the surface of the substrate, and in particular the linkermolecule layer. The barrier material is selectively applied by way of avariety of printing techniques to form exposed regions. A step ofdeprotection by way of deprotection agents may then be applied to theexposed regions. Preferably, the deprotection step occurs with use ofdeprotection agents in the vapor phase. This sequence of steps may beused for the selected synthesis of an array of oligonucleotides.

[0058] The present invention also provides for use of selected printingtechniques to apply deprotection agents, barrier materials, nucleosides,and the like for the synthesis of an array of oligonucleotides.Preferably, the type of printing technique should be able to transfer asufficient volume of print material to selected regions of the substratein an easy, accurate, and cost effective manner. Examples of variousprinting techniques for the synthesis of for example an array ofoligonucleotides are described herein. Further examples of theseembodiments of the present invention may be applied to the synthesis ofarrays of DNA as explained by application Ser. No. 07/796,243 in thename of Winkler et al., and U.S. Pat. No. 5,143,854 in the name ofPirrung et al., which are both hereby incorporated by reference for allpurposes.

[0059] Examples of suitable phosphoramidite synthesis methods aredescribed in the User Manual for Applied Biosystems Model 391, pp. 6-1to 6-24, available from Applied Biosystems, 850 Lincoln Center Dr.,Foster City, Calif. 94404, and are generally known by those skilled inthe art.

[0060] Chemical synthesis of polypeptides is known in the art and aredescribed further in Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501;Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al.(1989) Science 243: 187; Merrifield, B. (1986) Science 232: 342; Kent,S. B. H. (1988) Ann. Rev. Biochem. 57: 957; and Offord, R. E. (1980)Semisynthetic Proteins, Wiley Publishing, which are incorporated hereinby reference).

[0061] Once synthesized, polynucleotide arrays of the invention havemany art-recognized uses. For example and not limitation, thesynthesized sequences may be used as hybridization probes or PCRamplimers to detect the presence of a specific DNA or mRNA, for exampleto diagnose a disease characterized by the presence of an elevated mRNAlevel in cells, to identify a disease allele, or to perform tissuetyping (i.e., identify tissues characterized by the expression of aparticular mRNA), and the like. The sequences may also be used fordetecting genomic gene sequences in a DNA sample, such as for forensicDNA analysis (e.g., by RFLP analysis, PCR product length(s)distribution, etc.) or for diagnosis of diseases characterized byamplification and/or rearrangements of a characteristic gene.

EMBODIMENTS OF THE PRESENT INVENTION

[0062] An embodiment of the present invention may be briefly outlined byway of the following method.

[0063] 1. Provide a substrate.

[0064] 2. Optionally, form a layer of linker molecules on the substrate.

[0065] 3. Mechanically apply a barrier pattern on the linker moleculeswith exposed regions.

[0066] 4. Deprotect the linker molecules in the exposed regions withstandard DMT chemistry.

[0067] 5. Strip barrier pattern.

[0068] 6. Apply remaining synthesis steps.

[0069] This sequence of steps provides for an embodiment with use of abarrier layer with standard DMT chemistry. This provides for a desiredselectivity, easy in synthesis, low costs, high contrast, highresolution, among other features. Of course, this sequence of steps isshown for illustrative purposes only, and should not limit the scope ofthe appended claims herein.

[0070] An alternative embodiment of the present invention may be brieflyoutlined by way of the following method.

[0071] 1. Provide a substrate.

[0072] 2. Optionally, form a layer of linker molecules on the substrate.

[0073] 3. Selectively apply a print media by way of a printing technique(not a photosensitive printing techniques) on the linker molecules.

[0074] 4. Apply remaining synthesis steps.

[0075] This sequence of steps allows for the selective application of aprint medium onto a substrate by way of the various printing techniquesdescribed herein. These printing techniques simply do not use any exoticphotosensitive type materials, although later photosensitive steps canbe combined with the teachings herein. In preferred embodiments,deprotection agents may be introduced onto the substrate in vapor form.Accordingly, the present invention provides for the selectiveapplication of a variety of print media onto a substrate withoutnecessitating the use of conventional photosensitive materials.

[0076]FIG. 1 illustrates one embodiment according to the present method.A substrate 12 is shown in cross-section. The substrate may bebiological, nonbiological, organic, inorganic, or a combination of anyof these, existing as particles, strands, precipitates, gels, sheets,tubing, spheres, containers, capillaries, pads, slices, films, plates,slides, and the like. The substrate may have any convenient shape, suchas a disc, square, sphere, circle, etc. The substrate is preferably flatbut may take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whichthe synthesis takes place. The substrate and its surface preferably forma rigid support on which to carry out the reactions described herein.For instance, the substrate may be a functionalized glass, Si, Ge, GaAs,GaP, SiO₂, SiN₄, modified silicon, or any one of a wide variety of gelsor polymers such as (poly)tetrafluoroethylene, polypropylene,polyethylene, (poly)vinylidenedifluoride, poly-styrene, polycarbonate,or combinations thereof. Other substrate materials will be readilyapparent to those of skill in the art upon review of this disclosure. Ina preferred embodiment the substrate is flat glass or single-crystalsilicon with surface relief features of less than 10 microns. In anotherpreferred embodiment, the substrate is a polypropylene material.

[0077] Surfaces on the solid substrate will usually, though not always,be composed of the same material as the substrate. Thus, the surface maybe composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. In some embodiments the surface mayprovide for the use of caged binding members which are attached firmlyto the surface of the substrate. Preferably, the surface will containreactive groups, which could be carboxyl, amino, hydroxyl, or the like.Most preferably, the surface will have surface Si-OH functionalities,such as are found on silica surfaces. For synthesis of polynucleotidesby phosphoramidite chemistry, a linker consisting of(—COCH2CH2CONHCH2CH2CH2-siloxane bond-glass substrate) may be used toattach to a DMT-protected nucleoside via formation of a carboxyl bond tothe 3′ hydroxyl of the nucleoside.

[0078] The substrate 12 includes a surface 14 with a layer of linker (orspacer) molecules 16 thereon. The linker molecules are preferably ofsufficient length to permit polymers in a completed substrate tointeract freely with molecules exposed to the substrate. The linkermolecules should be about 4 to about 40 atoms long to provide sufficientexposure. The linker molecules may be, for example, aryl acetylene,ethylene glycol oligomers containing 2-10 monomer units, diamines,diacids, amino acids, among others, and combinations thereof.Alternatively, the linkers may be the same molecule type as that beingsynthesized (i.e., nascent polymers), such as oligonucleotides oroligopeptides.

[0079] In a preferred embodiment, the linker molecules are PEG linker.Of course, the type of linker molecules used depends upon the particularapplication.

[0080] The linker molecules can be attached to the substrate viacarbon-carbon bonds using, for example, (poly)trifluorochloroethylenesurfaces, or preferably, by siloxane bonds (using, for example, glass orsilicon oxide surfaces). Siloxane bonds with the surface of thesubstrate may be formed in one embodiment via reactions of linkermolecules bearing trichlorosilyl groups. The linker molecules mayoptionally be attached in an ordered array, i.e., as parts of the headgroups. In alternative embodiments, the linker molecules are absorbed tothe surface of the substrate.

[0081] The linker molecules or substrate itself and monomers used hereinare provided with a functional group to which is bound a protectivegroup. Preferably, the protective group is on the distal or terminal endof the linker molecule opposite the substrate. The protective group maybe either a negative protective group (i.e., the protective grouprenders the linker molecules less reactive with a monomer upon exposure)or a positive protective group (i.e., the protective group renders thelinker molecules more reactive with a monomer upon exposure). In thecase of negative protective groups an additional step of reactivationwill be required. In some embodiments, this will be done by heating.

[0082] In a subsequent step, the substrate 12 includes a barrier pattern18 with selected exposed regions 20 formed thereon. Each of the exposedregions corresponds to an “opening” in the barrier material where it isdesirable to remove protecting groups from the linker molecules. Theprotecting groups may be removed from the linker molecules by immersionin the deprotecting solution. Examples of the deprotecting solutioninclude trichloroacetic acid, hydrochloric acid, among others.

[0083] The barrier pattern can be made of any suitable material capableof masking certain regions of the linker molecule layer to protect suchregions from subsequent processing. The barrier pattern may include, forexample, materials such as a lacquer, an oil, a mask stencil, a siliconemask, an epoxy, a silicone oil, a polyester, a silicon membrane mask, aliquid capable of providing a barrier to protecting groups, a solidcapable of providing a barrier to protecting groups, among others, andcombinations thereof. The lacquer may include a lacquer such as Pactra63-1 and others, often having characteristics formulated to withstandhot fuel. An epoxy may include any suitable epoxy type material such asWest 105 and others. Selected oils are a rotary pump oil such as MowiocMC110, a silicone oil such as Dow Corning 704, and others. Polyestertype materials may include TAP SB and the like, and combinationsthereof.

[0084] The barrier pattern is applied as a liquid or a vapor by avariety of techniques. Examples of selected ways to apply the barriermaterial include brush, spray techniques, selected printing techniques,and others.

[0085] Selected printing techniques may be used for the application ofliquid barrier materials. The selected techniques of printing include arelief or letter press (the oldest form), gravure or intaglio, stencilprinting, lithography, among others. FIGS. 4-17, which will be describedin more detail below, illustrate a variety of printing techniques usedin applying the barrier material. Some of the techniques as applied inthe printing industry were from Printing Technology, J. Michael Adams,David D. Faux, Lloyd J. Ricker (3d.Ed., Delmar, 1988), which is herebyincorporated by reference for all purposes.

[0086] After optionally deprotecting the linker molecule layer, thebarrier material is then stripped by methods of wet chemical strip,acetone, IPA, and others. The linker molecule layer is then washed orotherwise contacted with a first monomer layer such as receptor “A” inFIG. 3. The first monomer reacts with the activated functional-groups ofthe linker molecules which have been deprotected. FIG. 3 illustrates asimplified cross-sectional diagram of the substrate 12, linker moleculelayer 12, and monomer layer “A.” The sequence of steps illustrated byFIGS. 1-3 may be repeated to achieve the desired sequence of monomers atselected regions to form an array of oligonucleotides, peptides, otherpolymers, and the like.

[0087] FIGS. 4-7 illustrate techniques of printing as relief or letterpress 50, gravure or intaglio 60, stencil printing 70, and lithography80, respectively. Relief printing 50 relies upon the use of raisedfeatures 52 to transfer printing medium to a substrate. As for Gravureor intaglio printing 60, it uses sunken features 62 to apply the desiredshape to a substrate. Stencil printing 70 which includes screenprinting, shadow masking, spray painting, and others, occurs throughmechanical openings 72 of a stencil 74. Lithography is a form ofprinting regions 82 of a surface that are chemically treated toselectively retain print medium. Each of these techniques may be usedfor the application of a barrier material, a carrier material, adeprotecting agent, or a polymer unit pattern onto a substrate.

[0088] More recent forms of printing include xerography (which includeslaser printing), ink jet printing (or print medium jet printing), andothers. FIGS. 8-13 illustrate the more recent forms of printing.

[0089]FIG. 8 illustrates a form of xerography printing 90. Xerographyprinting is directed to printing by way of an electrical charge pattern.Steps of xerography printing often include steps of charging 91,exposure 92, development 93, transfer 94, fixing 95, and cleaning 96.

[0090] FIGS. 9-10 illustrates two forms of printing known as ink jetprinting or in this case print medium jet printing. In this type ofprinting, print medium is forced through an array of orifices that isscanned across a workpiece, and is therefore really a form or derivativeof stencil printing. FIG. 9 illustrates a continuous ink jet process110. The continuous print medium jet process includes a substrate 111, acatcher assembly 112, a recycle 113, a deflector ribbon 114, a chargeplate 115, an orifice plate 116, among other elements. The print mediumjet type printer may deliver a pattern of selected print medium in asingle pass. A resolution of such printing technique can be as low asabout 200 microns and less.

[0091] The printhead includes a substrate 121, a heater assembly 122, abarrier 123, print medium 124, a nozzle plate 125, and the print mediumjet 126. The heater assembly 122 may be formed from a underlayer 127overlying the substrate, a resistive heater element 128 overlying theunderlayer, a conductor 129, and a overlying layer of passivation 130.The drop-on-demand printhead has the capability of delivering controlledamounts of fluids such as barrier medium, carrier material, monomerunits, and the like onto the surface of a workpiece.

[0092] A simplified cross-sectional view of an offset rotary press 140is illustrated by FIG. 11. The offset rotary press includes a impressioncylinder 141, a blanket cylinder 142, a plate cylinder 143, print mediumrollers 144, a solvent roller 145, and a substrate 146. The image istransferred (or offset) from the plate cylinder to the blanket cylinder,which reverses the image. The image is then passed to the presssubstrate (or sheet) as it moves between the blanket cylinder and theimpression cylinder.

[0093] Another form of offset printing such as gravure offset printing150 is illustrated by FIG. 12. Gravure offset printing includes steps ofprint medium transfer 151, transfer 152, and printing 153. A doctorblade 154 may be used to force medium 155 into grooves 158 during theprint medium transfer step. The print medium is then transferred onto ablanket cylinder 156, often covered with a rubber blanket 157. Theblanket cylinder then prints the print medium onto a substrate. Anexample of gravure offset printing is illustrated in Mikami et al., IEEETransactions on Electron Devices 41, 306, (March 1994), which is herebyincorporated by reference for all purposes. Mikami et al. applies offsetprinting for the manufacture of arrays of thin film transistors for flatpanel displays. By way of gravure offset printing, features sizes downto about 30 micrometers have been made.

[0094] Still a further printing technique 160 is illustrate by FIG. 13.The printing technique includes use of a plate cylinder 162, a pluralityof distribution rollers 164, a plurality of form rollers 166, a printmedium fountain roller 168, a ductor roller 169, and other elements.This type of technique provides for a more uniform distribution ofbarrier medium, carrier medium, deprotecting agents or polymer units (orconventional ink) in selected applications.

[0095] In preferred embodiments, spatially selectivity and in particularlocal selectivity is achieved by way of trapping liquid under anin-contact stencil mask. It should be noted that local selectivityrefers to the process of forming the liquid barrier at a selected orpredefined region. A liquid may attach to solid surfaces and pull themtogether with selected values of surface and interfacial energies. Thepull is characterized as a pressure P in the following relationship:

P=T/R

[0096] where

[0097] P is the suction pressure between the two surfaces;

[0098] T is the surface tension of the liquid; and

[0099] R is the radius of curvature of the meniscus.

[0100] The relationship assumes a small contact angle, but not so smallas to cause liquid to creep along surfaces of either solid. An exampleof this technique is illustrated in D. B. Tuckerman and R. F. W. Pease,Paper presented at U.S./Japan VLSI Symposium, Kaanapaali, Hi. 1983(Tuckerman et al.), which is hereby incorporated by reference for allpurposes. Tuckerman et al. discloses the use of DC 704 oil to attachintegrated circuit chips in this manner.

[0101] A suitable value for R often requires that the volume of liquidjust fills the gap between the solid surfaces of the barrier andsubstrate. Too little liquid and the gap empties to drain certainregions, thereby creating no deprotection at such regions. Too muchliquid corresponds to a larger R, and the reduction of the attractiveforce P. Of course, the selected amount of liquid depends upon theparticular application.

[0102]FIG. 14 illustrates a simplified cross-sectional view of anapparatus 170 used to achieve local selectivity. The apparatus includesa glass workpiece 172, a stencil 174, an upper electrode 176, a maskmount (or lower electrode) 178, and other features. The glass workpieceis positioned between the upper electrode and the mask mount. A voltagesuch as one of about 20,000 volts is applied to the upper electrode,while the mask mount (or lower electrode includes a potential at about 0volt. The difference in voltages provides an electrostatic force for theattachment of the workpiece to the stencil. An attractive pressurebetween the stencil and the workpiece ranges from about 0.5 gm-force/cm²to about 50 gm-force/cm² and is preferably about 5 gm-force/cm².

[0103] The attachment between the stencil and the workpiece allows fordeprotection agents to be introduced onto exposed regions 179 of theworkpiece. For example, a deprotection agent 177 such as vapor phase TCApasses over exposed regions of the workpiece, thereby causing selectivedeprotection of such regions.

[0104] In a preferred embodiment, the apparatus includes a jig 180 foraligning a stencil 181 coated with a liquid barrier material onto theworkpiece 183, as illustrated by FIG. 15. The jig substantially preventsthe liquid barrier material from smearing onto unselected regions ofcells on the workpiece. As shown, the jig includes a workpiece holder184, a stencil mount 185, sliding surfaces 186, among other features.The stencil mount includes a gasket 187, a stencil holder 188, thestencil mask 181, a spacer 189, sliding surfaces 186, and otherfeatures.

[0105] To align the coated stencil onto the workpiece, the coatedstencil is firmly placed onto the stencil holder of the stencil mount.The stencil mount then inserts into a cavity 191 of the workpieceholder. The coated stencil firmly abuts the surface of the workpiece.The tolerance between the sliding surfaces is about 1.0 micron to about10.0 microns, and is preferably at about 2.5 microns. An attractivepressure between the stencil and the workpiece ranges from about 0.8gm-force/cm² to about 50 gm-force/cm² and is preferably about 5gm-force/cm². A gasket made of a material such as teflon, polyisoprene,polymethyl methacrylate, and others is located between the spacer andthe stencil mask mount. This gasket absorbs shock between the stenciland workpiece to seal them together upon an applied pressure.

[0106] A further alternative embodiment provides a high resolutionstencil mask for ion beam proximity printing, ion beam projectionlithography, and the like. The high resolution stencil mask includes,for example, a silicon membrane mask, an epoxy barrier mask, and others.An other example of a high resolution stencil mask may includeelectroformed nickel, among others. A high resolution stencil maskincludes feature sizes of about 0.5 micron to about 50 microns, but lessthan about 100 microns. A high resolution stencil mask also includes athickness of about 2 to about 20 microns, and is preferably about 5microns and less. An example of such mask is made by Nanostructures,Inc.

[0107] The high resolution stencil mask attaches to the workpiece by theaforementioned techniques. For example, the stencil mask directlyattaches to the surface of the workpiece by placement. Alternatively,the stencil mask may be attached to the workpiece with an interfacialfluid. Further, the stencil mask attaches to the workpiece with use ofan electrostatic force, among other forces. The apparatus of FIGS. 14and 15 may also be used to attach the high resolution stencil onto theworkpiece. Of course, the type of high resolution stencil mask dependsupon the particular application.

[0108] In an alternative embodiment, the present invention provides forthe use of vapor phase deprotection agents. The vapor phase deprotectionagents may be introduced at low pressure, atmospheric pressure, amongothers. The use of such vapor phase deprotection agents allows for easein processing the work piece with linker molecules and barrier material.

[0109] Deprotection may be carried out in a directional stream ofdeprotection agent at low pressure. Low pressure deprotection occurs byfirst removing the work piece with linker molecules and barrier materialfrom the synthesizer. The work piece is then transferred into a vacuumchamber, preferably an extremely low pressure vacuum chamber. Thedeprotection agent is then bled into the vacuum chamber at a selectedrate to promote directionality of the deprotection agent stream.Preferably, the vacuum chamber includes pressures ranging from about10⁻⁵ torr to about 1 torr to promote the directionality of the stream.For example, the deprotection agents may include either a 2% solution ofTCA in DCP, solid TCA, among others, and combinations thereof. By use ofthe TCA/DCP solution or solid TCA, the vapor pressure ranges from about0.1 millitorr to about 1 torr to promote a directional stream ofdeprotection agents.

[0110] In a preferred embodiment, the deprotecting steps occur atatmospheric pressure. At atmospheric pressure, the work piece is heldover a solution of deprotection agents at atmospheric pressure, andpreferably at room temperature. An example of a deprotection agentincludes TCA, DCP, and the like, and combinations thereof. The TCA maybe mixed with DCP to form a 2% solution. Alternatively, the TCA may beused in pure form at room temperature, or at a temperature ranging fromabout 10° C. to about 50° C., and preferably at about 20° C. The workpiece is held over the TCA type deprotection agents for about 1 minuteor less. The TCA can also be blown against the workpiece by way offorced convection and the like, and even mixed with a water vapor, acarrier gas, or the like.

[0111] An advantage of the vapor phase deprotection agent, even atatmospheric pressure, is the lack of mechanical action to disturb anyphysical barrier pattern material. A further advantage with the vaporphase deprotection agent is the ease in use with selected work pieces.

EXAMPLES

[0112] 1. Use of Lacquer Barrier Material

[0113] An experiment was carried out with a standard 2 inch by 3 inchderivatized glass slide. An ABI Model 392 synthesizer was used to applyPEG linker-CC-DMT (linker layer-2 polymer units-protecting group) on oneside of the glass slide. The glass slide, also known as the work piece,was then removed from the reaction chamber of the synthesizer for theapplication of a barrier material.

[0114] To find a suitable barrier material, patterns of candidatematerials were applied with a fine paint brush to the linker layer. Thefine paint brush produced features made of the barrier material down toabout 100 microns. The barrier material was allowed to dry in air,typically at room temperature. The barrier material is intended toprovide an enclosure over selected regions of the linker layer. Theregions outside the selected regions were exposed for further processingsuch as a step of deprotection and further synthesis.

[0115] In this experiment, a lacquer known as Pactra 63-1 was found tobe an effective barrier material. A fine brush applied the lacquer asdots from about 0.1 mm to about 1 mm in dimension to the linker layer.The lacquer was dried for several hours at room temperature, beforesubsequent processing.

[0116] A step of deprotection followed the application of the barriermaterial. In this experiment, the workpiece was immersed into adeprotecting solution to remove the protecting group from the linkerlayer.

[0117] After deprotecting, the barrier material was stripped. In thisexperiment, stripping occurred by the use of acetone, gently wipedacross the lacquer. The use of acetone in this technique did not affectsubsequent coupling processes.

[0118] After stripping, the workpiece was reinserted back into thereaction chamber of the synthesizer. In the reaction chamber, theworkpiece was fluoreprime coupled. After removing the workpiece from thereaction chamber, a solution of ethylene diamine/ammonia was used toimmerse the workpiece for about 5 to 10 minutes.

[0119] The workpiece was then placed into a confocal scanningfluorescence microscope for inspection. The deprotected regionsfluoresced strongly, and the successfully shielded regions (or selectedregions) by the barrier material did not fluoresce. This experimentshows the effectiveness of the lacquer barrier material.

[0120] To achieve proper control over the experiment, the experimentaland control groups were synthesized and sampled as shown in Table 1.TABLE 1 Sample and Control Groups for Barrier Material FormationExperiment. # TYPE PROCESS RESULT 1 Control Apply barrier pattern,strip, No and scan. fluorescence. 2 Control Apply barrier pattern,strip, Complete fluoreprime, and scan, fluorescence. 3 Sample Applybarrier pattern, strip, Selective deprotect, fluoreprime, and scan.

[0121]FIG. 16 is a photograph of fluorescent image of the fluoreprimedworkpiece that was selectively shielded from liquid deprotection bylacquer. The photograph 200 shows a workpiece 202 with exposed regions204, and protected regions 205. As noted in Table 1, the layer ofbarrier material which includes the lacquer effectively shielded theprotecting groups, from deprotection. The contrast ratio between theexposed regions and the protected regions 205 is about 20:1.

[0122] From this experiment, it is concluded that at least one type ofmaterial such as the lacquer can serve as an effective barrier materialfor complete deprotection and complete protection. The lacquer barriermaterial is easily applied and dries at room temperature. It is alsoconcluded that at least one type of material such as acetone effectivelystrips the lacquer from the linker layer but does not affect thecoupling process.

[0123] 2. Use of Vapor-Phase Deprotection

[0124] Vapor phase deprotection was carried out on a standard workpiece.The workpiece was a standard 2 inch by 3 inch derivatized glass slide.An ABI Model 392 synthesizer was used to apply PEG linker-CC-DMT (linkerlayer-2 polymer units-protecting group) on one side of the glass slide.The glass slide also known as the work piece was fluoreprimed, asbefore. Experiments were performed at a variety of selected barriermaterials and deprotection agent pressures as follows.

[0125] A. Low Pressure Deprotection

[0126] To prove the principles of low pressure deprotection, anexperiment was carried out were a deprotection agent is introduced ontothe fluoreprimed workpiece at a vacuum. In this experiment, theworkpiece was removed from the synthesizer, and inserted into a vacuumchamber. The deprotection agent was bled in through a leak valve at aselected pressure, as measured by vacuum gauges. The vacuum gauges readpressures ranging from about 0.1 millitorr to about 1 torr. At thesepressures, the deprotection agent stream should be substantiallydirectional. Two forms of deprotection agents were used in thisexperiment as follows. TABLE 2 Low Pressure Deprotection Experiments #PROCESS RESULT 1 Apply 2% solution of TCA in DCP at Deprotectionpressures from about 0.1 millitorr to partially about 1 torr. completed.2 Apply TCA from vapor of solid TCA at Deprotection pressures from about0.1 millitorr to partially about 1 torr. completed.

[0127] From Table 2, it is clear that applying the deprotection agentsat low pressures provided for at least partial deprotection from thedeprotection agents. An advantage with the use of the deprotectionagents at low pressures is the directionality of the stream ofdeprotection agents. By controlling the directionality of thedeprotection agents, mechanical masks such as stencil masks may be usedas a barrier for subsequent processing without being in (intimate)contact with the workpiece.

[0128] B. Atmospheric Pressure Gas-Phase Deprotection

[0129] An experiment was also performed where the deprotection agentswere introduced onto the workpiece at atmospheric pressure. Theworkpieces were prepared as in the previous experiment at low pressure.A lacquer known as Pactra 63-1 was applied as dots from about 0.1 mm toabout 1 mm in dimension to the linker molecule layer of each of theworkpieces. The lacquer was dried for several hours at room temperature,before additional processing. The workpieces were then subjected todifferent deprotecting steps as shown in Table 3. TABLE 3 AtmosphericPressure Deprotection Experiments # PROCESS RESULT 1 Hold workpiece overa test tube Complete protection under containing a solution of about thelacquer dots and 2% TCA/DCP for about 60 partial deprotection seconds.elsewhere. 2 Hold workpiece over a vial Complete protection undercontaining solid TCA at about the lacquer dots and 20° C. for about 60seconds. complete deprotection elsewhere. 3 Hold workpiece over a vialPartial protection under containing solid TCA heated to the lacquer dotsand about 70° C. for about 60 complete deprotection seconds. elsewhere.The lacquer dots did not hold up to the hot TCA, which appeared tocondense on the workpiece.

[0130] Each of the samples 1, 2, and 3 was fluoreprimed afterdeprotection, and scanned to determine the effectiveness of thedeprotection agents. From the samples as noted in Table 3, the processof sample 2 has results that appear to be the most effective.Deprotection at room temperature (about 20° C.) with use of solid TCAprovided complete protection under the lacquer dots, and completedeprotection elsewhere, clearly desirable results.

[0131] C. Atmospheric Pressure Deprotection with Liquid BarrierMaterials

[0132] Atmospheric deprotection was also carried out using a variety ofdifferent barrier materials. These barrier materials include an epoxysuch as West 105 (an uncured epoxy resin), a rotary pump oil such asMowioc MC 110, a silicone oil such as Dow Corning 704, and a polyestersuch as TAP SB. The workpieces were made as before and the experimentswere carried out with the solid TCA deprotection agent at roomtemperature. Samples were held over the solid TCA for a period of about20 to about 60 seconds. All of the liquids appeared to act as effectivebarriers, that is, protection occurred underlying the liquid regions.The West 105 epoxy appeared to give the best results by having thecrispest edges, and was therefore chosen for later experiments asdescribed herein.

[0133]FIG. 17 is a photograph of dots of uncured epoxy and pump oiloverlying a workpiece. The photograph 300 shows a workpiece 302 withexposed regions 304, and protected regions 306, 308. As shown, theprotected regions include painted-on patches of uncured epoxy 306 (thepatches on the left-side of the dotted line), and patches of vacuum pumpoil 308 (the patches on the right-side of the dotted line). From thephotograph, the patches of uncured epoxy appeared to have crisper edgesthan the patches of vacuum pump oil. The contrast ratio for the uncuredepoxy was about 20:1.

[0134] D. Atmospheric Pressure Deprotection with Local Selectivity

[0135] Experiments were performed with use of the apparatus of FIG. 14and a liquid interface between a workpiece and a stencil mask. Thestencil mask was electroformed nickel, and coated uniformly with a layerof liquid barrier material such as an epoxy or a polyester. In thisexperiment, the liquid was prepared by dissolving the West 105 epoxymaterial in acetone. A solution of 0.1 ml. epoxy in 10 ml. acetone wasapplied to glass slide via a Sonotek ultrasonic spray nozzle. The glassslide was then spun at about 3000 revolutions per minute such that thesurface with solution is normal to the axis of revolution. Of course,the rotation speed and duration depends upon the desired film thicknessof remaining solution. The glass slide was then placed film side downonto the stencil mask. An electrode at about 22 kV on a back surface ofthe glass slide applies electrostatic force onto the workpiece,typically at about an attractive pressure of about 200 Pa (or about2×10⁻³ atmospheres or 2 grams-force/cm²). The glass slide is thenremoved from the mask which retains a coating of barrier material.

[0136] Alignment of the coated stencil mask with workpiece occurred withuse of the jig apparatus of FIG. 15. The jig apparatus brought thestencil mask in contact with the workpiece without smearing more than asmall fraction of a cell size, typically about 2.5 microns in thisexperiment. A mask using a 1,000 mesh/inch (25 micron pitch) grid wasused as a stencil mask.

[0137]FIG. 18 illustrates a SEM photograph 400 at 1300 timesmagnification of a liquid uncured epoxy pattern on a glass workpiece(following removal from stencil mask). As shown, the photograph includesa glass workpiece 402, a grid pattern of uncured epoxy 403, and exposedregions 404. The grid pattern bars are about 7 microns across. Thisphotograph demonstrates the accuracy of the aforementioned apparatus forthe placement of an epoxy barrier material onto a workpiece surfacewithout smearing.

[0138]FIG. 19 illustrates a SEM photograph 500 of a 100 micronresolution sample. The photograph 500 shows a workpiece 502 with exposedregions 504, and protected regions 506. The workpiece was prepared asbefore. A stencil mask made of nickel was coated with West 105 epoxy (anuncured epoxy resin) and electrostatically held against the workpiece.Each of the spaces between the protected regions was about 100 micronsin length from inner edge to inner edge. The contrast ratio between theexposed and protected regions was about 200:1. This photograph clearlyshows the effectiveness of present apparatus and West 105 epoxy. Itshould be noted that the arc shaped region 508 was caused by West 105epoxy applied with a fine paint brush.

[0139]FIG. 20 illustrates a SEM photograph of a 75 micron resolutionsample. The workpiece was prepared as before. The photograph 600 shows aworkpiece 602 with exposed regions 604, and protected regions 606. Theworkpiece was prepared as before. A stencil mask made of nickel wascoated with West 105 epoxy (an uncured epoxy resin) andelectrostatically held against the workpiece. Each of the spaces betweenthe protected regions is about 75 microns in length from inner edge toinner edge. The width of each of the bars is about 25 microns. Thecontrast ratio between the exposed and protected regions was less thanthat of FIG. 19. As even narrower width bars were attached to theworkpiece, the contrast ratio between the exposed and protected regionsdecreased. The photograph 600 shows a contrast ratio of about 4:1 toabout 2:1.

[0140] E. Atmospheric Pressure Deprotection Silicon Membrane Mask

[0141] An experiment was performed with high resolution silicon stencilmasks being fabricated for ion-beam proximity printing and ion beamprojection lithography. A mask available from Nanostructures, Inc. witha membrane thickness of about 2 to 4 microns was used. The mask wasmounted in openings of about 3 millimeter on an aluminum frame. Themasks structure with frame was electrostatically attached to theworkpiece without use of an interfacial fluid (because of risk ofrupturing the fragile membrane). The workpiece was made, as previouslynoted, in the manner described above. A step of vapor phase deprotectionoccurred on the workpiece with mask structure attached. The workpiecewas fluoreprimed and scanned, as the previous experiments. In thisexperiment, the results were encouraging, as illustrated in thephotograph of FIGS. 21 and 22.

[0142]FIG. 21 is the photograph of fluorescent pattern from the vapordeprotection through the uncoated silicon stencil mask. The photograph700 includes a workpiece 702 with exposed regions 704 and protectedregions 706. As noted, the protected regions were protected by way ofthe silicon mask. The fluorescent contrast ratios exceeded 10:1 forfeatures of about 50 microns, which are clearly desirable results. Nosubstantial “undercutting” (reaction under the stencil) occurred,presumably due to partial separation caused by particulatecontamination.

[0143] Other regions also suffered from undercutting, as can be seem bythe relative sizes of the arrowed features 708 in the FIG. 22photograph. The undercutting at such regions as defined by the arrowwere believed to be caused by particulate contamination preventing goodcontact. Of course, this problem may be cured, at least in part, byproper process controls, and the like.

[0144] In other experiments, less undercutting occurred for smallerfeatures such as about 5 microns and less. It is believed the smallerdimensions due to the smaller features had better contact betweenworkpiece and mask structure. The smaller features are more compliant sothe electrostatic force between mask and workpiece surface is moreeffective at maintaining good contact.

[0145] F. Atmospheric Pressure Deprotection with 105 Epoxy Barrier

[0146] Experiments were also performed to test the masking strength of105 epoxy manufactured by West, against vapor phase deprotection. Theworkpiece samples were prepared, as previously noted, same as theprevious experiments up to the deprotecting step. A film of 105 epoxywas spin coated to a thickness of about 0.8+/−0.3 microns (about 1milligram over about a 4×4 cm₂ surface). Thin fragments of silicon wereplaced at a region near the center of the field of view, and small markswere scored manually nearby, to create an unmasked region. The regionsunderlying the silicon film were completely masked, the scored regionscompletely unmasked, and the exposed regions of epoxy film were inquestion. After placement of such regions, the workpiece was air driedin a desiccator for about 24 hours, before the vapor phase deprotectionstep.

[0147] A vapor phase deprotection step was performed on the sampleworkpiece described above. The sample was then scanned. The fluorescentcount rate for each of the regions are listed in Table 4 below. TABLE 4Fluorescent Count Rate for 105 Epoxy Samples # REGION FLUORESCENT COUNTRATE 1 Region of exposed 105 epoxy on about 390 to about 490 theworkpiece. 2 Region of silicon fragments about 330 to about 390 coveringthe 105 epoxy. 3 Region of scored 105 epoxy to about 1530 to about 1630form unprotected area.

[0148] The Table 4 illustrates that thin film epoxy blocks at leastabout 75% of the deprotecting action, for example. Further tests mayneed to be performed to achieve higher contrasts, between protected andunprotected regions.

[0149] Another experiment was performed using a fine grid pattern of 105epoxy applied to a workpiece. The workpiece was made similar to thetechnique as described above up to the deprotecting step. A fine, clear,epoxy grid pattern of 25 microns half-pitch was applied to a centerregion of the workpiece. No epoxy was placed around such center region.Additional widths (down to about 25 microns) of epoxy were applied withan ultra-fine paint brush.

[0150] After about 24 hours of air drying in a desiccator, the workpieceunder went a step of vapor phase deprotection. A scanned fluorescentimage was uniformly bright except for the center region masked by theepoxy pattern. In the masked region, the grid pattern was visible butcontrast and brightness were lower. Signal strength in the nominallyclear regions of the grid pattern was significantly lower than that fromregions more than about 100 microns from the epoxy pattern. Accordingly,it appears as if the West 105 material inhibits the deprotection withinregions of about 100 microns of such material.

[0151] Other experiments were performed using finer marks of the 105epoxy, an epoxy paint (TAP “Copon” Clear), a polyester resin (TAPplastics), and a Dow Corning 704 silicone oil. At low magnification theresults were as before, that is, the fluorescent image of the oil markshad poor acuity, but the polyester and epoxy matter were fine. However,at high magnification (resolution at about 100 microns and less) the 105epoxy did have less sharp of an edge than either the epoxy paid or thepolyester marks. In particular, the epoxy paint pattern showed the 1micron pixel size of the scanner, and was therefore determined thepreferred barrier material.

[0152] G. Atmospheric Pressure Deprotection With TAP “Copon” Clear Paint

[0153] In the experiment with use of epoxy paint, printed patterns ofthe TAP “Copon” clear paint were formed by painting on a solution of 1:4(paint:spray thinner) on to a 500 mesh/inch nickel grid (Buckbee Mears).The grid with fresh paint was then electrostatically attached to theworkpiece, transferring the paint pattern on the surface of theworkpiece. The grid was then removed from the surface of the workpiece,leaving the paint pattern behind.

[0154]FIG. 23 is a photograph 800 of the paint pattern transferred fromthe nickel grid. The photograph 800 includes a workpiece 802 with anepoxy paint pattern 804 and exposed regions 806. The distance in each ofthe exposed regions is about 25 microns, and the pattern includes bars,each having a width of about 20 microns. This type of printing is anexample of high resolution gravure printing, as described above. Thetransferred paint pattern acted as the barrier material duringsubsequent deprotecting steps. As shown, the paint patterns were crisp(and fine lined) to create an effective mask for printing a barrierpattern to obtain a diverse array of oligonucleotides.

[0155]FIGS. 24 and 25 are photographs of fluorescent images resultingfrom vapor phase deprotection through an epoxy pattern, similar to theone shown by the photograph of FIG. 23. Both of the photograph show goodcontrast ratios between the exposed and protected regions. Thephotographs do not show any visible proximity effect and the contrastratio exceeds 10:1, clearly desirable results.

[0156] 3. Hybridization Experiments

[0157] To demonstrate the effectiveness of the aforementioned techniqueson the synthesis of oligonucleotides, selected experiments wereperformed. 2×2 arrays of oligonucleotides were prepared on substrates1002 using silicon fragments (pieces of silicon material), which wereelectrostatically attached as crude masks at base #4 (A) and #5 (T).FIG. 26 illustrates a 2×2 array 1000 of oligonucleotides formed bymasking out the deprotect agents after A (vertical mask 1009) and thefirst T in the synthesis of 3′-CGCATTCCG 1004. A 10 nM target5′-GCGTAGGC-fluorescein at 15 C was exposed in the flow cell to thearray, which was then scanned with a scanner. The four probes were3′-CGCATCCG (match) 1005, 3′-CGCTCCG (deletion) 1006, 3′-CGCTTCCG(substitution) 1007, and 3′-CGCATTCCG (addition) 1004.

[0158]FIG. 27 is a photograph of a representative scanned fluorescentoutput, which shows the counts obtained in the four areas. The matchedarea is the most strongly fluorescing, indicating the strongesthybridization to the match and the weakest to the deletion. The whitespots appear most frequently in the matching region and was acharacteristic of all experiments using this particular target. Anothersample was hybridized with a more freshly prepared target oligo andobtained better results (as in FIG. 28) that were further enhanced byhybridizing at 10° C. for 15 minutes and then scanning at 15° C. Thecounts in the matching region 1005 were more than doubled with only amodest increase in the unmatched regions as shown in FIG. 29.

[0159] Because the synthesis of the probes, with the exception of theaddition, involved at least one removal from the synthesizer for maskeddeprotection, control experiments were performed forming the other threeprobes as uninterrupted sequences on the reverse side of three separatesubstrates. These sequences were hybridized and scanned to check for anysignificant difference in counts between those grown uninterruptedly onthe reverse side with those grown on the front using the masking. Therewere no significant differences with those grown on the front using themasking. Hence we conclude that one or two interruptions for maskedvapor-phase deprotection introduces no significant undesiredperturbation of the growth of the probes.

[0160] A further check was made on an array 2000 of four oligos of FIG.26) by moving the reaction chamber around the substrate at bases 4 and 5as shown by FIG. 31 to compare directly the behavior of a (simple) arraymade with vapor-phase deprotection at one or two bases with theconventional ABI chemistry throughout. The results are shown in FIGS. 28and 29. The results were similar to those obtained with the array madewith vapor-phase deprotection including the enhanced selectivity andsignal from the matching area following hybridization at 10 C for 15minutes. Thus no detectable difference between the results obtained withvapor-phase deprotection with those made conventionally were seen.

[0161] While the above is a full description of the specificembodiments, various modifications, alternative constructions, andequivalents may be used. For example, while the description above is interms of the synthesis of oligonucleotide arrays, it would be. possibleto implement the present invention with peptides, small molecules, otherpolymers, or the like. Alternatively, the embodiments may also be incontext to peptides, other polymers, or the like.

[0162] Therefore, the above description and illustrations should not betaken as limiting the scope of the present invention which is defined bythe appended claims.

What is claimed is:
 1. A method of forming polymers having diversemonomer sequences on a substrate, said method comprising: providing asubstrate comprising a linker molecule layer thereon, said linkermolecule layer comprising a linker molecule and a protective group;applying a barrier layer overlying said linker molecule layer, saidapplying step forming selected exposed regions of said linker moleculelayer; and exposing said selected exposed regions of said linkermolecule layer to a vapor comprising a deprotecting agent.
 2. The methodof claim 1 wherein said deprotection agent is an acidic vapor selectedfrom a group consisting of TCA, DCA, and HCl.
 3. The method of claim 1wherein said deprotection agent is at a temperature ranging from about20° C. to about 50° C.
 4. The method of claim 1 wherein said applyingstep is selected from a group consisting of relief press printing,letter press printing, gravure printing, intaglio printing, stencilprinting, and lithography.
 5. The method of claim 1 wherein said barrierlayer is selected from a group consisting of a lacquer, an epoxy, anoil, a polyester, and a polyurethane.
 6. The method of claim 1 whereinsaid barrier layer is a liquid.
 7. The method of claim 1 wherein saiddeprotection agent comprises a carrier gas.
 8. The method of claim 1wherein said deprotection agent comprises a water vapor.
 9. A method ofdeprotecting selected regions of a substrate, said method comprising:providing a substrate comprising a layer of linker molecules thereon,each of said linker molecules having a protective group; applying avapor deprotection agent to selected regions of said linker moleculelayer.
 10. The method of claim 9 wherein said deprotection agent isselected from a group consisting of TCA, DCA, and HCl.
 11. The method ofclaim 9 wherein said deprotection agent is at a temperature ranging fromabout 20° C. to about 50° C.
 12. The method of claim 9 wherein saidapplying step occurs through forced convention.
 13. The method of claim9 wherein said deprotection agent comprises a carrier gas.
 14. Themethod of claim 9 wherein said deprotection agent comprises a watervapor.
 15. A method of applying a medium in selected regions of asubstrate, said method comprising the steps of: providing a partiallycompleted substrate comprising an array of monomers, said partiallycompleted substrate having a top surface; selectively applying a mediumcomprising an element selected from a group consisting of a barriermaterial, a receptor, a deprotection agent, a monomer group, a carriermaterial, and an activator to selected regions of said substrate topsurface.
 16. The method of claim 15 wherein said medium is selected froma group consisting of TCA, DCA, HCL, and any other acidic vapor.
 17. Themethod of claim 15 wherein said medium is at a temperature ranging fromabout 20° C. to about 50° C.
 18. The method of claim 15 wherein saidmedium is a deprotection agent.
 19. The method of claim 18 wherein saiddeprotection agent comprises a carrier gas.
 20. The method of claim 18wherein said deprotection agent comprises a water vapor.
 21. The methodof claim 15 wherein said selectively applying step is selected from agroup consisting of relief press printing, letter press printing,gravure printing, intaglio printing, and stencil printing.
 22. Themethod of claim 15 wherein said selectively applying step occurs througha drop-on-demand printhead.
 23. A method of synthesizing anoligonucleotide comprising the steps of: coupling a first portion ofsaid oligonucleotide to said substrate, said first portion of saidoligonucleotide comprising a removable protecting group; removing saidprotecting group with a vapor phase deprotection agent to expose afunctional group on said first portion of said oligonucleotide; andcovalently bonding a second portion of said oligonucleotide to saidfirst portion of said oligonucleotide.
 24. The method as recited inclaim 23 wherein said surface of said substrate is selectively protectedby a mask during said removing step.
 25. The method as recited in claim24 further comprising repeating said removing and covalently bondingsteps to form an array of oligonucleotides.
 26. The method of claim 24wherein said vapor phase deprotection agent is selected from a groupconsisting of TCA, DCA, and HCl.
 27. The method of claim 24 wherein saidmask is selected from a group consisting of an epoxy, a silicone oil, ametal, a silicon material, a lacquer, a oil, and a polyester.
 28. Themethod of claim 24 wherein said mask is held in place by electrostaticforce.